Viruses are the major causative agents of central nervous system (CNS) infection worldwide. DNA viruses such as herpes simplex virus-1 (HSV-1) trigger broad activation of glial cells including microglia and astrocytes, eliciting the release of an array of mediators that can promote innate and adaptive immune responses. Such responses can limit viral replication and dissemination leading to infection resolution. However, a defining feature of viral CNS infection is the rapid onset of severe neuroinflammation, and overzealous glial responses are associated with significant neurological damage or even death. The mechanisms by which microglia and astrocytes perceive neurotropic viruses are only now becoming apparent with the discovery of a variety of cell surface and cytosolic molecules that serve as sensors for viral components. DNA-dependent activator of interferon- regulatory factors (DAI) was the first identified sensor of cytosolic double-stranded DNA (dsDNA) that can elicit innate immune responses and induce type I IFN to control viral replication. DAI has been shown to mediate interferon production and inflammatory cytokine expression by transfected murine cell lines in response to intracellular viral DNA and HSV-1. Importantly, our published preliminary studies indicate that both microglia and astrocytes functionally express this putative viral sensor and we have used siRNA knockdown techniques to show that DAI plays a significant role in the in vitro responses of these CNS cell types to HSV-1 infection. In this pilot R03 study, we will employ isolated primary microglia and astrocytes derived from mice genetically deficient in the expression of this novel cytosolic viral sensor (DAI-/-) to confirm the functional expression of DAI by these cells, and to demonstrate its importance in in vitro inflammatory and anti-viral responses to the neurotropic DNA virus, HSV-1. Furthermore, we will employ DAI-/- animals to begin to evaluate the in vivo role of this viral sensor in DNA virus control and neuropathology in our established mouse model of acute HSV-1 infection. The proposed pilot R03 studies will provide critical preliminary data regarding the importance of DAI in the initiation of immune responses within the CNS during DNA virus infection, and will add greatly to our understanding of the events that trigger the development of either protective host responses or damaging neuroinflammation. In addition, these pilot studies will provide a solid rationale for a more comprehensive investigation into the regulation and role of this novel viral pattern recognition receptor in CNS infection for which future R01 mechanism support will be sought.